Abstract: Human pluripotent stem cells (hPSCs) are promising resources as cell-based therapies for the debilitating injuries caused by many neuro-degenerative diseases. However, controlling hPSC differentiation into lineage-specific neural cells is one of the most important problems needed to be addressed before their potential for neuro-regenerative medicine can be fully realized. A detailed insight into the functions of extracellular microenvironments and intrinsic cellular regulators which dynamically regulate the hPSC neurogenesis into neural/neuronal cells is a prerequisite for addressing the aforementioned challenges. However, functions of hPSC microenvironments are much more complicated to investigate because of our lack of knowledge about the multiple signals inducing differentiation and limited methods available for investigation. Therefore, the primary focus of our study is to develop innovative methods to identify optimal cues for hPSC differentiation into subtype specific neurons and genetic manipulation of hPSCs using non-viral siRNA based transfection tools. Our innovative approaches will allow for the establishment of novel cell-based assay tools and siRNA based genetic manipulation tools for selective and efficient neuro-differentiation of hPSCs. Moreover, efforts will be made to integrate these studies into one multianalytic microfluidics platform for synchronized control of microenvironmental cues and intrinsic cellular regulators synergistically. The PI's research experiences in nanoscale biomaterials, functional genomics, and stem cell biology and current interdisciplinary research programs aiming at investigating cellular interactions within microenvironments would be critical to develop the aforementioned/innovative tools. Public Health Relevance: Neuro-degenerative diseases (e.g. Alzheimer's disease and Parkinson's disease) and spinal cord injury, affects about a few million Americans, who experience life-long debilitating paralysis or even death due to the injury. Therefore, there is an urgent need for the development of cell-based therapies for neuro-regenerative medicine, where human pluripotent stem cells (hPSCs) are extremely promising resources for transplantation therapies as they possess the unique ability to self-renew and give rise to all somatic cell lineages. Goals of this proposed study is to develop innovative methods for identifying as well as understanding the temporal/spatial effects from microenvironmental cues and intrinsic cellular programs on growth, differentiation, and molecular specification of human pluripotent stem cell (hPSC) differentiation into sub-type specific neurons.